Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-06-18
2004-07-27
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C549S220000, C549S221000
Reexamination Certificate
active
06767716
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to enhanced chemiluminescence of 1,2-dioxetane compounds, derived from the oxidation of novel alkenes prepared by the coupling of substituted aromatic esters or ketones and spiro-fused ketones with or without a &pgr;-electron system or a carbon—carbon double bond, in the presence of water-insoluble or partially water-soluble or water-soluble polymers obtained from natural or synthetic sources. More particularly, the present invention relates to a 1,2-dioxetane chemiluminescent system which can achieve a detection level as low as an attogram of enzyme or “single molecule” detection. Even more particularly, the present invention relates to a 1,2-dioxetane chemiluminescent system which includes (1) a stabilized dioxetane which can be destabilized from alkaline phosphatase or other enzymes and emit light on decomposition, (2) an enhancer for enhancing the light intensity to a maximum limit, and (3) an enzyme diluent or buffer that stabilizes the enzyme for extended periods of time during the reaction and does not effect the efficiency of the enhancer and the 1,2-dioxetane, and methods of use thereafter
2. Prior Art
Chemiluminescence and bioluminescence have been tested as potential replacements for radioactive labels, not only in competitive and sandwich-type immunoassays but, also, in DNA sequencing and other related research. Chemiluminescence is advantageous also for detecting and measuring trace elements and pollutants for environmental control. Thus, research on the chemiluminescence of organic compounds is an on-going area of major emphasis. Chemiluminescence provides a major advantage since it generates cold light, i.e. its generated light is not caused by vibrations of atoms and/or molecules involved in the reaction, but by direct transformation of chemicals into electronic energy. The best known chemiluminescent reactants are stabilized 1,2-dioxetanes, acridanes, acridinium esters, luminol, isoluminol and derivatives thereof, and lucigenen.
Stabilized 1,2-Dioxetanes
Chemiluminescent compounds, their preparation and their uses have been long documented in the prior art. These “high energy” molecules store sufficient energy to generate, on fragmentation, electronically excited carbonyl products which are responsible for the observed chemiluminescence. Dioxetanes and, especially, 1,2-dioxetanes are eminently useful to detect the presence, as well as the absence, of certain enzymes in fluids such as blood and the like because of their chemiluminescence. Thus, 1,2-dioxetanes are eminently useful in doing medical assays.
Generally, 1,2-dioxetanes are thermally labile substances having a wide range of stability and which decompose on heating to emit light according to the following reaction:
where each R corresponds to any one of a multitude of organic moieties widely reported in the prior art, as detailed herebelow. As noted these 1,2-dioxetanes have a wide range of stability. For example, the prior art, as found in: (a) K. W. Lee, L. A. Singer and K. D. Legg, J.Org.Chem., 41, 2685(1976); (b) F. McCapra, I. Beheshti, A. Burford, R. A. Hanu and K A. Zakika, J.Chem.Soc., Chem.Commun.,944(1977); and (c) J. H. Wieringa, J. Strating, H. Wynberg and W. Adam, Tet. Lett.,169 (1972); respectively, disclose the following 1,2-dioxetanes of different stability:
Although these high energy compounds are all spiro-substituted 1,2-dioxetanes. Spiroadamantane substitution exerts a tremendous stabilizing effect on these four-membered ring peroxides. The lower activation energy (EA) of the dioxetanes of formulae (3) and (4) is explained by the donation of charge from nitrogen to the dioxetane ring. The dioxetane of formula (5) decomposes at 150° C. and has a half-life, at 25° C., of more than 20 years.
A stabilized 1,2-dioxetane (6) below, dispiro[adamantane-2,3′-[1,2-dioxetane]-4,9-fluorene] was isolated as crystals and described by W. Adam and L. A. A. Encarnacion, Chem. Ber.,115, 2592 (1982).
The stability of 1,2-dioxetanes (5) and (6) is described on the basis of the bulky and rigid spiro nature of the adamantane group.
The first stable and enzymatic triggerable 1,2-dioxetane was synthesised by the oxidation of (6-acetoxy-2-naphthyl) methoxy methyleneadamantane as reported by A. P. Schaap, R. S. Handley and B. P. Giri, Tet. Lett., 935 (1987). This 1,2-dioxetane utilizes aryl esterase emzyme to catalyze the cleavage of the acetate group of a naphthylacetate-substituted-1,2-dioxetane and produce chemiluminescence in aqueous buffers at ambient temperature by the following sequence:
Several other stabilized 1,2-dioxetanes and their use as enzyme substrates have been disclosed in the literature. See, inter alia, A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva and B. P. Giri, Tet. Lett., 1155(1987); A. P. Schaap, M. D. Sandison and R. S. Handley, Tet. Lett., 1159 (1987); U.S. Pat. No. 4,962,192; U.S. Pat. No. 4,978,614; U.S. Pat. No. 5,386,017; U.S. Pat. No. 5,721,370, other useful 1,2-dixetanes are described in the copending patent apply, the disclosures of which are hereby incorporated by reference.
These several other 1,2-dioxetanes, generally, have the following general structures:
wherein
is a non-active site, i.e. non-reactive and which is selected from the group of polycyclic alkyl groups containing 6 to 30 carbon atoms, OX is an oxy group substituted on an aryl ring which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent, and X is a chemically labile group which is removed by the activating agent to form light and carbonyl containing compounds, R
1
is a lower alkyl containing 1 to 8 carbon atoms, or mixtures thereof, or
wherein T is a non-active site which is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is hydrogen, alkyl, aryl arylkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme cleavable group, and Z is hydrogen or an enzyme cleavable group, provided that at least one of X or Z must be an enzyme cleavable group, or
wherein,
(1) when Ar—O—Y and OR join together to give an aryl group substituted with an O—Y group to form a stable 1,2-dioxetane intermediate which is triggerable to form an unstable intermediate oxide, R
2
and R
3
either form (a)
which is either cyclic, polycyclic or a spiro-fused ring containing at least one carbon—carbon double bond or cabon—carbon triple bond in the ring or side chain with or without hetero atoms, or (b)
which is either cyclic, polycyclic or a spiro-fused ring containing a substituted or unsubstituted fused aromatic ring or substituted or unsubstituted aromatic ring attached by linker arms;
(2) when Ar—O—Y and OR
1
do not join together
a) Ar is an aryl which may be phenyl, substituted phenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl and the like as well as a nonaromatic fluorescent or nonfluorescent group; Y is hydrogen, alkyl, acetate, t-butyldimethylsilyl or an enzyme or a group of enzymes cleavable group, or an antibody cleavable group; R
1
is selected from the group consisting of alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)
2
, alkyl(etheralkyl)
3
, alkyletherhaloalkyl, alkyl(etherhaloalkyl)
2
, alkylalkene, alkylalkyne, arylalkene, arylalkyne, linear or branched halogenated alkyl, alkylalcohol, alkylnitrile, alkylamine, alkylacid or an inorganic salt thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid or an inorganic salt, thereof, linker-flourescent molecule, linker-antibodies, linker-antigen, linker-biotin, linker-avidin, linker-protein, linker-carbohydrates or linker-lipids; R
2
and R
3
form either (i)
which is cyclic, polycyclic or a spiro-fused ring containing at least one carbon—carbon double bond or cabon—carbon triple bond in the ring or side chain with or without heteroatoms, or (ii)
Gitomer Ralph
The Weintraub Group P.L.C.
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